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Wolbachia infection in native populations of Blattella germanica and Periplaneta americana

  • Nayyereh Choubdar,

    Roles Formal analysis, Investigation, Methodology, Software, Writing – original draft

    Affiliation Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

  • Fateh Karimian,

    Roles Data curation, Formal analysis, Methodology, Software

    Affiliation Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran

  • Mona Koosha,

    Roles Conceptualization, Data curation, Formal analysis, Methodology, Software

    Affiliation Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

  • Jalil Nejati,

    Roles Investigation, Methodology

    Affiliation Health Promotion Research Center, Zahedan University of Medical Sciences, Zahedan, Iran

  • Razieh Shabani Kordshouli,

    Roles Investigation, Methodology

    Affiliation Department of Medical Entomology and Vector Control, Health Sciences Research Center, School of Public Health, Mazandaran University of Medical Sciences, Sari, Iran

  • Amrollah Azarm,

    Roles Investigation, Methodology

    Affiliation Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran

  • Mohammad Ali Oshaghi

    Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – review & editing,

    Affiliation Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran


Cockroaches are significant pests worldwide, being important in medical, veterinary, and public health fields. Control of cockroaches is difficult because they have robust reproductive ability and high adaptability and are resistant to many insecticides. Wolbachia is an endosymbiont bacterium that infects the reproductive organs of approximately 70% of insect species and has become a promising biological agent for controlling insect pests. However, limited data on the presence or strain typing of Wolbachia in cockroaches are available. PCR amplification and sequencing of the wsp and gltA genes were used to study the presence, prevalence and molecular typing of Wolbachia in two main cockroach species, Blattella germanica (German cockroach) and Periplaneta americana (American cockroach), from different geographical locations of Iran. The Wolbachia endosymbiont was found only in 20.6% of German cockroaches while it was absent in American cockroach samples. Blast search and phylogenetic analysis revealed that the Wolbachia strain found in the German cockroach belongs to Wolbachia supergroup F. Further studies should investigate the symbiotic role of Wolbachia in cockroaches and determine whether lack of Wolbachia infection may increase this insect’s ability to tolerate or acquire various pathogens. Results of our study provide a foundation for continued work on interactions between cockroaches, bacterial endosymbionts, and pathogens.


Cockroaches have survived on Earth for more than 300 million years, virtually unchanged. They are one of the most successful groups of animals because of their adaptability to various environmental conditions [1]. There are about 4500 species of cockroaches in the world, among which about 30 species frequently cohabit with human populations, and a few are indoor pests that are important in medical, veterinary, and public health fields [2, 3]. The indoor health pests prefer humid, dark, and dirty environments, where they are exposed to various microorganisms [46].

As an ideal biological vector, the cockroaches can acquire and mechanically transmit diverse human pathogens, including bacteria, fungi, and parasites, thereby causing serious human diseases [4, 710]. German cockroaches Blattella germanica Linnaeus 1767, (Blattodea: Blattidae) and American cockroaches Periplaneta americana Linnaeus 1758, (Blattodea: Blattidae) are, respectively, the first and second most contaminated cockroach species that threaten human health [11]. At the same time, cockroaches are an important source of allergens, and their faeces, debris, and secretions may cause severe allergic reactions, such as allergic asthma [1214]. All cockroaches, but particularly the German and American cockroaches, which live in close contact with human, are also important reservoirs and transmission vectors of antibiotic resistance bacteria and/or antibiotic resistance genes [1517].

In recent years, many studies have shown that cockroaches are rich in microbes [3, 4, 16, 18], and some of these microbes have formed an interdependent symbiotic relationship with the host during the long-term co-evolution process. Wolbachia is a gram-negative, maternally inherited, intracellular Rickettsia-like alphaproteobacterium that is found in many arthropods, such as insects, mites, ticks, arachnids, terrestrial crustaceans (Isopods) and filarial nematodes [19]. This bacterium is one of the most successful organisms on earth and is found globally, infecting from 20–76% of all insects [20, 21]. The bacterium acts as a reproductive parasite in arthropods where it manipulates its host’s reproduction capacity, causing a variety of abnormalities including cytoplasmic incompatibility (CI), feminization of genetic males (FM), male killing (MK) and thelytokous parthenogenesis (TP) [2224].

It is known that bacterial endosymbionts may provide their host with nutritional benefit or with protection against xenobiotics, parasites and infections, insecticides, natural enemies, and abiotic stresses [25]. For example, Wolbachia produces B vitamins to support bed bugs [26, 27], or could support fly development and buffers against nutritional stress resulted in reduced pupal mortality, increased adult emergence, and larger size in several fly genotypes [28, 29]. It is suggested that Wolbachia acts as a nutritional symbiont to supplement insect development and increase host fitness: a selective advantage that could promote to its high occurrence in nature [28]. Certain endosymbionts may also alter acquisition and transmission of pathogens particularly arboviruses by insect vectors [30]. Wolbachia has been testified to suppress a variety of pathogen infections in Aedes, Anopheles, or Culex mosquitoes by either competition for limited nutrients or induced host immune responses [3137].

Although Wolbachia is widely reported in many groups of insects, but so far limited studies on Wolbachia in cockroaches have been carried out. Wolbachia infections have been reported in a few cockroaches such as Blattella sp. and Supella longipalpa [38]. The present study was carried out to detect the presence, infection rate, distribution, and phylogeny of naturally acquired Wolbachia infection in cockroach populations of B. germanica and P. americana in Iran.

Materials and methods

Ethics statement

The protocols were conducted in this study followed the guidelines of the institutional ethical committee (Tehran University of Medical Sciences, TUMS). The protocols were approved by TUMS ethical committee under registry IR.TUMS.SPH.REC.1400.112.

Specimen collection and morphological identification

Cockroach specimens were collected from 13 sampling sites across the country between April and November 2021 (Fig 1, Table 1).

Fig 1. Map of sampling sites for B. germanica and P. americana across Iran.

Numbers are 1: Ardabil, 2: West Azarbaijan, 3: Ilam, 4: Khuzestan, 5: Kohgiluye and Boyer-Ahmad, 6: Fars, 7: Kerman, 8: Yazd, 9: Isfahan, 10: Tehran, 11: Mazandaran, 12: Razavi Khorasan, 13: Sistan and Baluchestan. Reprinted from Choubdar et al 2021 under a CC BY license, with permission from PLOS publisher, original copyright. (

Table 1. Details of cockroach specimens collected in this study.

Two cockroach species (P. americana and B. germanica) were collected and stored in 50ml centrifuge tubes and delivered to the laboratory of insect molecular biology, School of Public Health, Tehran University of Medical Sciences. The cockroaches were identified using relevant taxonomic keys and descriptions [39]. A subset of individuals from each species and location was selected and preserved at –80°C for subsequent molecular investigation.

Tissue dissection

The legs and wings of specimens were removed before isolating the gut and reproductive tissues. Tissue-specific dissection was carried out on each sample and dissected tissues, including the reproductive organs and alimentary canal, of each specimen were individually placed into small Petri dish in diameter size of 35 mm on ice to prevent DNA degradation. All dissection equipment and microscope slides were thoroughly wiped with 70% ethanol before commencing dissection of each sample.

DNA extraction and PCR amplification

DNA was extracted from each specimen using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, and the extracted DNA was stored at minus 20°C. In this study, two molecular markers, Wolbachia surface protein (wsp) and the citrate synthase (gltA) genes, were used to detect Wolbachia infection. The wsp general primers were 81F (5′-TGGTCCAATAAGTGATGAAGAAAC-3′) and 691R (5′-AAAAATTAAACGCTACTCCA-3′) [40] and the gltA primers, were WgltAF1 (5′-TACGATCCAGGGTTTGTTTCTAC-3′) and WgltARev2 (5′-CATTTCATACCACTGGGCAA-3′) [41] and these served to confirm Wolbachia identity.

For the wsp gene amplification, extracted DNA samples from the dissected tissues were screened by PCR in a thermocycler (Eppendorf, Hamburg, Germany) using the following protocol: initial denaturation at 95°C for 4 min; 35 cycles of 94°C 1min, 55°C 1min and 72°C 1min and final elongation step of 72°C for 10 min. For the gltA gene amplification, initial denaturation at 95°C for 2 min; two cycles of denaturation at 95°C for 2 min, annealing at 60°C for 1 min and extension at 72°C for 1 min; 35 cycles of denaturation at 95°C for 30s, annealing at 60°C for 1 min and extension at 72°C for 45s; final extension at 72°C for 10 min.

All PCR procedures were performed in reaction mixtures consisting of 12.5μl of Taq DNA Polymerase Master Mix RED (Denmark), 3μl of extracted DNA, and 1μl each of 5μM forward and reverse primers for Wolbachia PCR screens. Double-distilled water was used to top up the reaction mixture to a final volume of 25μl. PCR amplification of positive and negative controls was also conducted simultaneously. The negative controls were prepared with ddH2O and DNA from a male Anopheles stephensi mosquito; a positive control was prepared in our laboratory using DNA extracted from Drosophila melanogaster which harbours Wolbachia wMelPop strain. Amplicons were separated by gel electrophoresis on 1.5% agarose gel stained with green viewer (Parstous, Iran) and visualised under an ultraviolet transilluminator.

Gene sequencing and phylogenetic analysis

The criteria set to confirm Wolbachia infection were based on successful amplification of the molecular markers. Furthermore, samples that met this criterion were sequenced by bidirectional sequencing at Genomin Sequencing Centre, Iran. A subset of the PCR products of wsp and gltA gene representatives of different locations were purified from gels using a gel purification kit and subjected to sequencing. Sequencing was performed using an ABI 3730 sequencer machine. The resultant sequences were checked to correct ambiguities. Homologies with the available sequence data in GenBank were checked by using basic local alignment search tool (BLAST) analysis software ( A subset of consensus sequences for both loci was deposited in the GenBank database (Table 2). A subset of the available representative Wolbachia wsp and gltA sequences of nine supergroups (A-G, M, and T for wsp gene and A-F, and H-K for gltA gene) were acquired from public databases (http://www.ncbi.nlm) for phylogenetic analysis (Table 3). Owing to the different lengths of these sequences, all those used for alignment were trimmed to obtain consistent regions that were 443 bp for wsp and 599 bp for gltA genes, respectively. Multiple alignments of the sequences were carried out using the Clustal W algorithm in software MEGA X [42]. The short sequence reads were excluded. Pairwise sequence divergence, using Kimura’s 2-parameter distance algorithm, and neighbour joining (NJ) tree, shown in Figs 2 and 3, were processed in MEGA X. The robustness of all phylogenetic trees was tested with a bootstrapping value in NJ. The wsp sequence of Anaplasma centrale (Genbank ID: AB211162) and gltA sequence of Bartonella quintana (Genbank ID number: M73228) were acquired from Genbank and used as outgroups for each gene (Table 2).

Fig 2.

Phylogenetic relationship inferred from 443 bp of wsp gene sequences of B. germanica from Iran (OM928504-OM928515 that are shown by black square) and some representative sequences of Wolbachia strains belonging to supergroups A, B, C, D, E, F, G, and M obtained from Genbank database. The Genbank ID numbers, the host species, and the Wolbachia supergroup are shown in the tree branches respectively. The phylogenetic tree was created using Neighbour Joining–Kimura. Scale bar shows genetic distance. Numbers in the tree represent the bootstrap value (bootstrap values below 50% are not shown at the nodes).

Fig 3.

Phylogenetic relationship inferred from 599 bp of gltA gene sequences of B. germanica from Iran (OP146461-OP146466 that are shown by black circles) and some representative sequences of Wolbachia strains belonged to supergroups A, B, C, D, E, F, H, I, J, and K obtained from Genbank database. The Genbank ID numbers, the host species, and the Wolbachia supergroup are shown in the tree branches respectively. The phylogenetic tree was created using Neighbour Joining–Kimura. Scale bar shows genetic distance. Numbers in the tree represent the bootstrap value (bootstrap values below 50% are not shown at the nodes).

Table 2. Representative wsp and gltA Wolbachia type sequences used in this study.

Table 3. Prevalence of Wolbachia infection in cockroaches collected from different localities of Iran.

Data analysis

Statistical significance was determined as P < 0.05. All statistical analyses were performed in SPSS statistics version 21.


Wolbachia detection and prevalence

A total of 965 cockroaches, representing two species, were collected from 13 localities in Iran (Fig 1). PCR assays, using the primers described in Materials and Methods, gave the expected amplification products of 632 bp and 659 bp, for wsp and gltA respectively. Overall, only B. germanica was found to host Wolbachia. The other cockroach species (P. americana) showed no PCR amplification products. From a total of 544 German cockroaches screened by wsp marker, 95 specimens (17.46%) were found to be infected with Wolbachia (Table 3). The wsp-negative samples were re-evaluated with the gltA gene primers, and 17 out of 449 wsp-negative samples were found to be gltA-positive; thus, the totals number of Wolbachia-infected German cockroaches was 112 out of 544 (20.6%). The prevalence of Wolbachia infection was almost twice as high in females as in males (59 versus 32), which is statistically significant (P<0.001).

The Blast search of the wsp and gltA sequences of infected B. germanica revealed a high homology, with the F supergroup of Wolbachia strains found in Cimex lectularius (AP013028), Blattella sp (DQ354917), and Supella longipalpa (EF193198) (Figs 2 and 3). Following Vaishampayan et al’s finding, the wsp sequences obtained in this study can be classified into Wolbachia F supergroup [36]. According to our knowledge, this is the first report of Wolbachia infection in B. germanica in Iran (Table 3).

The sequences generated during this study have been deposited in the Gen Bank database (OM928504-OM928515 for wsp and OP146461-OP146467 for gltA sequences).

Phylogenetic analysis

The neighbour joining phylogenetic tree between the Wolbachia strain identified in this study and other known available Wolbachia strains are shown in Figs 2 and 3. Fig 2 shows a phylogenetic tree inferred from 433 bp of wsp gene sequences from Iranian isolates of B. germanica and some representative sequences of Wolbachia strains belonging to supergroups A, B, C, D, E, F, G, and M obtained from Genbank database. The phylogenetic analysis showed that Wolbachia strains identified from the Iranian specimens were closely associated and clustered with the Wolbachia strains presented in Blattella sp (DQ354917) and Supella longipalpa (EF193198) of supergroup F (Fig 2). In addition, in the phylogenetic analysis of gltA sequences, independent of the method for tree reconstruction, the cockroach Wolbachia sequences from the Iranian German cockroaches clustered with supergroup F (Fig 3) and led to similar tree topologies as found with the wsp gene. Phylogenetic analysis of the Wolbachia sequences with Maximum likelihood (ML), Neighbour Joining (NJ) and Maximum Parsimony (MP) methods showed almost similar topology.


In the current research we found that only about twenty percent of B. germanica specimens collected from the 13 provinces of Iran were positive for Wolbachia. This result concurs with the study of Vaishampayan et al in India, which reported that 20% of B. germanica examined harboured Wolbachia [38]. Also, we found no Wolbachia infection in American cockroaches. The low rate of Wolbachia infection in the German cockroaches and the lack of infection in the American cockroaches may indicate a lack of dependence, or a very low dependence, of the cockroach species on the endosymbiont for their survival and reproduction, in what is known as obligatory relationships [27, 56, 57]. Therefore, the low level of Wolbachia infection in cockroaches negates the possibility of Wolbachia being an obligatory endosymbiont in these cockroach species. Also, it is possibility that Wolbachia may have some negative effects on the fitness of cockroaches, which would counteract the possibility of Wolbachia expanding in the population through providing fitness advantages.

The low rate or lack of Wolbachia infection in the cockroaches may change in future because it is shown that changing the gut microbiota composition with antibiotic treatment enhanced Wolbachia density in Drosophila melanogaster [58]. We have no evidence to assess the impact of antibiotic treatment on the incidence and frequency of Wolbachia in German cockroaches, however, cockroaches are exposed to antibiotics in places such hospitals [59, 60] and most bacterial agents isolated from cockroaches are antidrug-resistant and antibiotic-resistant [61]. These situations provide cockroaches with diverse antibiotic treatments which may result in raising Wolbachia density in future and could be the subject of future studies.

The Wolbachia strain found in German cockroaches in this study belongs to supergroup F, which is consistent with previous studies indicating supergroup F in other cockroaches, such as Supella longipalpa and Blattella sp [38]. Wolbachia supergroup F has also been detected in bedbugs and nematodes. The Wolbachia supergroup F is essential for the bedbugs’ growth and reproduction because the bacterium provides B vitamins, which are deficient in their blood-based diet [27]. Therefore, the Wolbachia strain might promote persistence by providing fitness advantages to the German cockroach via nutrient supplementation [27, 62]. However, we do not yet know if this strain could provide any nutrient supplement for German cockroaches, and this needs to be determined by further studies.

Wolbachia strains are divided into 20 supergroups, ranging from A to U (G was not considered anymore) which diverged around 100 million years ago, first in filarial nematodes and then infecting arthropods [63]. In this study we found F supergroup in the cockroaches which is also found in distantly related host species including nematodes and domestic indoor pests (Cimex, Supella and Blattella). Wolbachia is transmitted either vertically between host generations or horizontally to other individuals and species through a mechanism called host shift (HS) [6365].

German or American cockroaches are omnivorous synanthropic insects, frequently encountering high loads of diverse microbes, and are reservoirs and vectors of several pathogens particularly pathogenic bacteria [61, 6668]. For example, Dokor showed that about a quarter of the microorganisms isolated from cockroaches are food-borne pathogens including Escherichia coli O157:H7, Staphylococcus aureus, Bacillus cereus, Shigella dysenteriae, Salmonella enterica subsp. enterica serovar Typhi, Rotavirus, Aspergillus fumigatus, and Cryptosporidium parvum [66]. Although Wolbachia has been shown to protect insects from a range of microbial and eukaryotic pathogens including viruses, Plasmodium and filarial nematodes [3137], there is no strong evidence that Wolbachia-infected insects can be protected against pathogenic bacteria. In an experiment, no difference in mortality was observed in the Drosophila simulans lines with five different Wolbachia strains or without Wolbachia when the lines were challenged with the pathogenic bacteria. Similarly, no antibacterial protection or upregulation of the antibacterial immune genes was observed for D. melanogaster infected with Wolbachia compared to paired flies without Wolbachia. It was suggested that Wolbachia-mediated antibacterial protection is not universal in insects and furthermore that the mechanisms of antibacterial and antiviral protection are independent [69]. In another study it was found that D. melanogaster flies harbouring no endosymbionts, those carrying both Spiroplasma and Wolbachia, and those containing Wolbachia only had parallel survival rates following infection with the virulent insect pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli bacteria [70]. Also, Wolbachia presence did not provide a protective advantage against entomopathogenic fungi, Beauveria bassiana and B. brongniartii, in two important mosquito vectors, Aedes albopictus and Culex pipiens that naturally carry Wolbachia [71]. Taken together these results plus high microbial loads, we suggest that presence of Wolbachia supergroup F may not provide protection in the German cockroach species. Also, having found Wolbachia supergroup F in German cockroaches warrants further studies to determine if Wolbachia supergroup F can manipulate the cockroach’s reproduction system through such means as cytoplasmic incompatibility (CI), induction of parthenogenesis (IP), male-killing (MK), or feminization of genetic males (MF) [46, 7274].

In this study the cockroaches were molecularly screened for Wolbachia DNA using two primer sets targeting partial wsp and gltA genes. Comparing the results of this study pointed the discrepancy in results between the primer pairs where Wolbachia DNA detected at the wsp locus was less than at the gltA locus. Baldo et al [75] showed that wsp gene has a mosaic structure with four hypervariable regions (HVRs), which provide a reasonable explanation for the negative results. It is possible that there is a trade-off between sensitivity and specificity of primer sets and certain primer sets can be more efficient than others, but that no single protocol can ensure the specific detection of all known Wolbachia infections [76].

In the present study we found that there was a sex bias toward infection in females of B. germanica, with Wolbachia prevalence in females being higher than in males. Wolbachia infections tend to confer reproductive advantages on their female hosts, which is the sex responsible for vertical transmission of the bacteria from one generation to another, leading to increased prevalence and propagation within the host populations [74]. Higher Wolbachia infection in females has already been reported in few hosts, such as hard ticks [77], fruit flies [78], and fleas [79]. Low levels of Wolbachia infection in male cockroaches also tends to support rejection of the possibility of Wolbachia as an obligatory endosymbiont, at least in males. Finally, the low occurrence of Wolbachia in male cockroaches may suggests the absence of robust CI in German cockroaches.


In conclusion, we found low or no Wolbachia infection in German and American cockroaches, respectively, calling for additional surveys of hidden fitness, nutrition, or protection properties, the reproductive manipulation such as CI occurrence, and underlying systems with sex-bias differences in Wolbachia persistence. Nevertheless, the long evolutionary history of Wolbachia’s interaction with invertebrate hosts and its adaptations for germ line transmission contribute to the value of Wolbachia for control of insect pests.


The manuscript was edited by the ICGEB Editing service (


  1. 1. Atiokeng Tatang RJ, Tsila HG, Wabo Poné J. Medically important parasites carried by cockroaches in Melong Subdivision, Littoral, Cameroon. J Parasitol Res. 2017; 3:1–8. pmid:28912965
  2. 2. Beccaloni G, Eggleton P. Taxonomy of blattodea. Zootaxa. 2011; 3148(1):199–200.
  3. 3. Pan X, Wang X, Zhang F. New insights into cockroach control: using functional diversity of Blattella germanica symbionts. Insects. 2020; 11(10):696. pmid:33066069
  4. 4. Akbari S, Oshaghi MA, Hashemi-Aghdam SS, Hajikhani S, Oshaghi G, Shirazi MH. Aerobic bacterial community of American cockroach Periplaneta americana, a step toward finding suitable paratransgenesis candidates. J Arthropod Borne Dis. 2015;9(1): 35–48. PMCID: PMC4478416.
  5. 5. Hashemi-Aghdam SS, Oshaghi MA. A checklist of Iranian cockroaches (Blattodea) with description of Polyphaga sp as a new species in Iran. J Arthropod Borne Dis. 2015; 9(2):161–175. PMCID: PMC4662788.
  6. 6. Yang CL, Zhu HY, Zhang F. Comparative proteomics analysis between the short-term stress and long-term adaptation of the Blattella germanica (Blattodea: Blattellidae) in response to beta-cypermethrin. J Econ Entomol. 2019; 112(3):1396–1402. pmid:30835785
  7. 7. Hashemi-Aghdam SS, Rafie G, Akbari S, Oshaghi MA. Utility of mtDNA-COI barcode region for phylogenetic relationship and diagnosis of five common pest cockroaches. J Arthropod. Borne Dis. 2017; 11(12):182–193. PMCID: PMC5641607. pmid:29062843
  8. 8. Kassiri H, Zarrin M, Veys-Behbahani R. Pathogenic fungal species associated with digestive system of Periplaneta americana (Blattaria: Blattidae) trapped from residential dwellings in Ahvaz city, southwestern Iran. J Arthropod Borne Dis. 2018; 12(1):16–23. PMCID: PMC6046110.
  9. 9. Salehzadeh A, Tavacol P, Mahjub H. Bacterial, fungal and parasitic contamination of cockroaches in public hospitals of Hamadan, Iran. J Vector Borne Dis. 2007; 44(2):105–110. pmid:17722863
  10. 10. Wannigama DL, Dwivedi R, Zahraei-Ramazani A. Prevalence and antibiotic resistance of gram-negative pathogenic bacteria species isolated from Periplaneta americana and Blattella germanica in Varanasi, India. J Arthropod Borne Dis. 2014; 8(1):10–20. PMCID: PMC4289503.
  11. 11. Nasirian H. Contamination of cockroaches (Insecta: Blattaria) by medically important bacteria: a systematic review and meta-analysis. J Med Entomol. 2019; 56(6):1534–1554. pmid:31219601
  12. 12. Kalayci O, Miligkos M, Beltrán CFP, El-Sayed ZA, Gómez RM, Hossny E, et al. The role of environmental allergen control in the management of asthma. World Allergy Organ J. 2022; 15(3):e100634. pmid:35341023
  13. 13. Sheih A, Parks WC, Ziegler SF. GM-CSF produced by the airway epithelium is required for sensitization to cockroach allergen. Mucosal Immunol. 2017; 10(3):705–715. pmid:27731325
  14. 14. Vazirianzadeh B, Mehdinejad M, Dehghani R. Identification of bacteria which possible transmitted by Polyphaga aegyptica (Blattodea: Blattidae) in the region of Ahvaz, SW Iran. Jundishapur J Microbiol. 2009; 2(1): 36–40.
  15. 15. Akinjogunla OJ, Odeyemi AT, Udoinyang EP. Cockroaches (Periplaneta americana and Blattella germanica): reservoirs of multi drug resistant (MDR) bacteria in Uyo, Akwa Ibom State. J. Biol. Sci. 2012;1(2):269–279.
  16. 16. Latorre A, Domínguez-Santos R, García-Ferris C, Gil R. Of cockroaches and symbionts: recent advances in the characterization of the relationship between Blattella germanica and its dual symbiotic system. Life. 2022; 12(2):290. pmid:35207577
  17. 17. Pai HH. Multidrug resistant bacteria isolated from cockroaches in long-term care facilities and nursing homes. Acta Trop. 2013; 125(1):18–22. pmid:22960645
  18. 18. Domínguez-Santos R, Pérez-Cobas AE, Cuti P, Pérez-Brocal V, García-Ferris C, Moya A, et al. Interkingdom gut microbiome and resistome of the cockroach Blattella germanica. Msystems. 2021; 6(3):1213–20. pmid:33975971
  19. 19. Zug R, Hammerstein P. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PloS One. 2012; 7(6):e38544. pmid:22685581
  20. 20. Hilgenboecker K, Hammerstein P, Schlattmann P, Telschow A, Werren JH. How many species are infected with Wolbachia? a statistical analysis of current data. FEMS Microbiol Lett. 2008; 281(2):215–220. pmid:18312577
  21. 21. Jeyaprakash A, Hoy MA. Long PCR improves Wolbachia DNA amplification: wsp sequences found in 76% of sixty‐three arthropod species. Insect Mol Biol. 2000; 9(4):393–405. pmid:10971717
  22. 22. Rigaud T. Feminizing endocytobiosis in the terrestrial crustacean Armadillidium vulgare Latr.(Isopoda): recent acquisitions. Endocyto Cell Res. 1991; 7: 259–273.
  23. 23. Stouthamer R, Breeuwer JAJ, Luck RF, Werren, JH. Molecular identification of microorganisms associated with parthenogenesis. Nature. 1993; 361(6407):66–68. pmid:7538198
  24. 24. Yen JH, Barr AR. New hypothesis of the cause of cytoplasmic incompatibility in Culex pipiens L. Nature. 1971; 232(5313): 657–658. pmid:4937405
  25. 25. Kaur R, Shropshire JD, Cross KL, Leigh B, Mansueto AJ, Stewart V, et al. Living in the endosymbiotic world of Wolbachia: a centennial review. Cell Host Microbe. 2021; 29(6):879–93. pmid:33945798
  26. 26. Zug R, Hammerstein P. Bad guys turned nice? A critical assessment of Wolbachia mutualisms in arthropod hosts. Biol. Rev. 2015; 90(1):89–111.
  27. 27. Hosokawa T, Koga R, Kikuchi Y, Meng XY, Fukatsu T. Wolbachia as a bacteriocyte-associated nutritional mutualist. Proc Natl Acad Sci USA. 2010; 107(2):769–74. pmid:20080750
  28. 28. Lindsey AR, Parish AJ, Newton IL, Tennessen J, Jones MW, Stark N. Wolbachia is a nutritional symbiont. bioRxiv. 2023:2023–01.
  29. 29. Newton IL, Rice DW. The Jekyll and Hyde symbiont: could Wolbachia be a nutritional mutualist?. Journal of bacteriology. 2020; 202(4):e00589–19. pmid:31659008
  30. 30. Lindsey AR, Bhattacharya T, Newton IL, Hardy RW. Conflict in the intracellular lives of endosymbionts and viruses: a mechanistic look at Wolbachia-mediated pathogen-blocking. Viruses. 2018; 10(4):141. pmid:29561780
  31. 31. Zhang D, Wang Y, He K, Yang Q, Gong M, Ji M, et al. Wolbachia limits pathogen infections through induction of host innate immune responses. PloS One. 2020; 15(2):e0226736. pmid:32078642
  32. 32. Caragata EP, Dutra HL, Moreira LA. Exploiting intimate relationships: controlling mosquito-transmitted disease with Wolbachia. Trends Parasitol. 2016; 32(3):207–218. pmid:26776329
  33. 33. Moreira LA, Iturbe-Ormaetxe I, Jeffery JA, Lu G, Pyke AT, Hedges LM, et al. A Wolbachia symbiont in Aedes aegypti limits infection with dengue, Chikungunya, and Plasmodium. Cell. 2009; 139(7):1268–78. pmid:20064373
  34. 34. Van Den Hurk AF, Hall-Mendelin S, Pyke AT, Frentiu FD, McElroy K, Day A, et al. Impact of Wolbachia on infection with chikungunya and yellow fever viruses in the mosquito vector Aedes aegypti. PLOS Negl Trop Dis. 2012; 6(11):e1892. pmid:23133693
  35. 35. Dutra HL, Rocha MN, Dias FB, Mansur SB, Caragata EP, Moreira LA. Wolbachia blocks currently circulating Zika virus isolates in Brazilian Aedes aegypti mosquitoes. Cell host & microbe. 2016; 19(6):771–4. pmid:27156023
  36. 36. Bian G, Joshi D, Dong Y, Lu P, Zhou G, Pan X, et al. Wolbachia invades Anopheles stephensi populations and induces refractoriness to Plasmodium infection. Science. 2013; 340(6133):748–51. pmid:23661760
  37. 37. Micieli MV, Glaser RL. Somatic Wolbachia (Rickettsiales: Rickettsiaceae) levels in Culex quinquefasciatus and Culex pipiens (Diptera: Culicidae) and resistance to West Nile virus infection. J Med Entomol. 2014; 51(1):189–99. pmid:24605469
  38. 38. Vaishampayan PA, Dhotre DP, Gupta RP, Lalwani P, Ghate H, Patole MS, et al. Molecular evidence and phylogenetic a affiliations of Wolbachia in cockroaches. Mol Phylogenet Evol. 2007; 44:1346–1351. pmid:17350292
  39. 39. Choate P, Burns S, Olsen L, Richman D, Perez O, Patnaude M, et al. A dichotomous key for the identification of the cockroach fauna (Insecta: Blattaria) of Florida. Fla Entomol. 2008; 72(4):612–617.
  40. 40. Zhou W, Rousset F, O’Neill S. Phylogeny and PCR–based classification of Wolbachia strains using wsp gene sequences. Proceedings of the Royal Society of London. Series B: Biol. Sci. 1998; 265(1395): 509–515. pmid:9569669
  41. 41. Casiraghi M, Bordenstein SR, Baldo L, Lo N, Beninati T, Wernegreen JJ, et al. Phylogeny of Wolbachia pipientis based on gltA, groEL and ftsZ gene sequences: clustering of arthropod and nematode symbionts in the F supergroup, and evidence for further diversity in the Wolbachia tree. Microbiology. 2005; 151(12):4015–4022.
  42. 42. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35(6):1547–1549. pmid:29722887
  43. 43. Karami M, Moosa-Kazemi SH, Oshaghi MA, Vatandoost H, Sedaghat MM, Rajabnia R, et al. Wolbachia endobacteria in natural populations of Culex pipiens of Iran and its phylogenetic congruence. J Arthropod Borne Dis. 2016; 10(3):347–363. PMCID: PMC4906741.
  44. 44. Bazzocchi C, Jamnongluk W, O’neill SL, Anderson TJ, Genchi C, Bandi C. wsp gene sequences from the Wolbachia of filarial nematodes. Curr Microbiol. 2000; 41(2):96–100. pmid:10856373
  45. 45. Lamb TJ, Le Goff L, Kurniawan A, Guiliano DB, Fenn K, Blaxter ML, et al. Most of the response elicited against Wolbachia surface protein in filarial nematode infection is due to the infective larval stage. J Infect Dis. 2004; 189(1):120–127. pmid:14702162
  46. 46. Ma Y, Chen WJ, Li ZH, Zhang F, Gao Y, Luan YX. Revisiting the phylogeny of Wolbachia in Collembola. Ecol. Evol. 2017; 7(7):2009–2017. pmid:28405268
  47. 47. Nikoh N, Hosokawa T, Moriyama M, Oshima K, Hattori M, Fukatsu T. Evolutionary origin of insect–Wolbachia nutritional mutualism. Proc Natl Acad Sci. 2014; 111(28):10257–10262. pmid:24982177
  48. 48. Rowley SM, Raven RJ, McGraw EA. Wolbachia pipientis in Australian spiders. Curr Microbiol. 2004; 49(3): 208–214. pmid:15386106
  49. 49. Augustinos AA, Santos-Garcia D, Dionyssopoulou E, Moreira M, Papapanagiotou A, Scarvelakis M, et al. Detection and characterization of Wolbachia infections in natural populations of aphids: is the hidden diversity fully unravelled? PloS One. 2011; 6(12):28695. pmid:22174869
  50. 50. Laidoudi Y, Levasseur A, Medkour H, Maaloum M, Ben Khedher M, Sambou M, et al. An earliest endosymbiont, Wolbachia massiliensis sp. nov., strain PL13 from the bed bug (Cimex hemipterus), type strain of a new supergroup T. Int. J. Mol. Sci. 2020; 21(21): 8064. pmid:33138055
  51. 51. Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A, et al. Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl Environ Microbiol. 2006; 72(2):1102–1109.
  52. 52. Paraskevopoulos C, Bordenstein SR, Wernegreen JJ, Werren JH, Bourtzis K. Toward a Wolbachia multilocus sequence typing system: discrimination of Wolbachia strains present in Drosophila species. Curr Microbiol. 2006; 53(5): 388–395. pmid:17036209
  53. 53. Ros VI, Fleming VM, Feil EJ, Breeuwer JA. How diverse is the genus Wolbachia? Multiple-gene sequencing reveals a putatively new Wolbachia supergroup recovered from spider mites (Acari: Tetranychidae). Appl Environ Microbiol. 2009; 75(4):1036–1043. pmid:19098217
  54. 54. Bordenstein S, Rosengaus RB. Discovery of a novel Wolbachia supergroup in Isoptera. Curr Microbiol. 2005; 51(6):393–398. pmid:16252129
  55. 55. Anderson BE, Dawson JE, Jones DC, Wilson KH. Ehrlichia chaffeensis, a new species associated with human ehrlichiosis. J Clin Microbiol. 1991; 29(12): 2838–2842.
  56. 56. Ferrari J, Vavre F. Bacterial symbionts in insects or the story of communities affecting communities. Philos Trans R Soc Lond B Biol Sci. 2011; 366:1389–1400. pmid:21444313
  57. 57. Moran NA, McCutcheon JP, Nakabachi A. Genomics and evolution of heritable bacterial symbionts. Annu. Rev. Genet. 2008; 42:165–190. pmid:18983256
  58. 58. Yixin HY, Seleznev A, Flores HA, Woolfit M, McGraw EA. Gut microbiota in Drosophila melanogaster interacts with Wolbachia but does not contribute to Wolbachia-mediated antiviral protection. J. Invertebr. Pathol. 2017; 143:18–25. pmid:27871813
  59. 59. Laborda P, Sanz-García F, Ochoa-Sánchez LE, Gil-Gil T, Hernando-Amado S, Martínez JL. Wildlife and antibiotic resistance. Front. cell. infect. 2022; 12: 873989. pmid:35646736
  60. 60. Donkor E.S. Nosocomial pathogens: an in-depth analysis of the vectorial potential of cockroaches. Trop. Med. Infect. Dis. 2019; 4(1):14. pmid:30658473
  61. 61. Molewa ML, Barnard T, Naicker N. A potential role of cockroaches in the transmission of pathogenic bacteria with antibiotic resistance: A scoping review. J. Infect. Dev. Ctries. 2022; 16(11):1671–8. pmid:36449637
  62. 62. Brownlie JC, Cass BN, Riegler M, Witsenburg JJ, Iturbe-Ormaetxe I, McGraw EA, et al. Evidence for metabolic provisioning by a common invertebrate endosymbiont, Wolbachia pipientis, during periods of nutritional stress. PLoS Pathog. 2009; 5(4):1000368. pmid:19343208
  63. 63. Gomes TM, Wallau GL, Loreto EL. Multiple long-range host shifts of major Wolbachia supergroups infecting arthropods. Sci. Rep. 2022; 12(1):8131. pmid:35581290
  64. 64. Siozios S, Gerth M, Griffin JS, Hurst GD. Symbiosis: Wolbachia host shifts in the fast lane. Curr. Biol. 2018; 28(6):R269–71. pmid:29558644
  65. 65. Pimentel AC, Beraldo CS, Cogni R. Host-shift as the cause of emerging infectious diseases: Experimental approaches using Drosophila-virus interactions. Genet. Mol. Biol. 2020; 44. pmid:33237151
  66. 66. Donkor E.S. Cockroaches and food-borne pathogens. Environ. Health Insights. 2020: 1178630220913365. pmid:32425541
  67. 67. Rahpaya SS, Tsuchiaka S, Kishimoto M, Oba M, Katayama Y, Nunomura Y, et al. Dembo polymerase chain reaction technique for detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs. J. Vet. Sci. 2018; 19(3):350–7. pmid:29284216
  68. 68. Moges F, Eshetie S, Endris M, Huruy K, Muluye D, Feleke T, et al. Cockroaches as a source of high bacterial pathogens with multidrug resistant strains in Gondar town, Ethiopia. Biomed Res. Int. 2016; 2016:2825056. pmid:27340653
  69. 69. Wong ZS, Hedges LM, Brownlie JC, Johnson KN. Wolbachia-mediated antibacterial protection and immune gene regulation in Drosophila. PloS one. 2011; 6(9):e25430. pmid:21980455
  70. 70. Shokal U, Yadav S, Atri J, Accetta J, Kenney E, Banks K, et al. Effects of co-occurring Wolbachia and Spiroplasma endosymbionts on the Drosophila immune response against insect pathogenic and non-pathogenic bacteria. BMC microbiol. 2016; 16(1):1–3. pmid:26862076
  71. 71. Ramirez JL, Schumacher MK, Ower G, Palmquist DE, Juliano SA. Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections. PLOS Negl Trop. Dis. 2021; 15(11):e0009984. pmid:34843477
  72. 72. Hurst GD, Jiggins FM. Male-killing bacteria in insects: mechanisms, incidence, and implications. Emerg Infect Dis. 2000; 6(4):329–336. pmid:10905965
  73. 73. Narita S, Kageyama D, Hiroki M, Sanpei T, Hashimoto S, Kamitoh T, Kato Y. Wolbachia‐induced feminisation newly found in Eurema hecabe, a sibling species of Eurema mandarina (Lepidoptera: Pieridae). Ecol. Entomol. 2011; 36(3): 309–317.
  74. 74. Werren JH, Baldo L, Clark ME. Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol. 2008; 6(10):741–751. pmid:18794912
  75. 75. Baldo L, Lo N, Werren JH. Mosaic nature of the Wolbachia surface protein. J. Bacteriol. 2005; 187(15):5406–18.
  76. 76. Simões PM, Mialdea G, Reiss D, Sagot MF, Charlat S. Wolbachia detection: an assessment of standard PCR protocols. Mol. Ecol. Resour. 2011; 11(3):567–72. pmid:21481216
  77. 77. Lo N, Beninati T, Sassera D, Bouman EA, Santagati S, Gern L, et al. Widespread distribution and high prevalence of an alpha‐proteobacterial symbiont in the tick Ixodes ricinus. Environ. Microbiol. 2006; 8(7):1280–1287.
  78. 78. Richardson KM, Griffin PC, Lee SF, Ross PA, Endersby-Harshman NM, Schiffer M, et al. A Wolbachia infection from Drosophila that causes cytoplasmic incompatibility despite low prevalence and densities in males. Heredity. 2019; 122(4): 428–440. pmid:30139962
  79. 79. Flatau R, Segoli M, Hawlena H. Wolbachia endosymbionts of fleas occur in all females but rarely in males and do not show evidence of obligatory relationships, fitness effects, or sex-distorting manipulations. Front Microbiol. 2021; 12: e649248. pmid:33776981